Cancer-relevant signal transduction involves a large number of signaling proteins with many parallel steps and interconnected feedback mechanisms. These properties of signal transduction processes cannot readily be measured by current techniques, which limits researchers ability to validate each of the more than 10,000 human signaling proteins as potential drug targets for cancer therapy. Over the last several years, my laboratory has made three separate developments that can now be integrated into a technology to systematically characterize complex signal transduction networks. In this approach, cell arrays will be made for simultaneously monitoring a large number of signaling processes. The proposed Evanescent Wave Cell Array Technology (ECAT) incorporates: 1) a microvolume electroporation method for RNA transfection, 2) a set of single cell GFP-tagged biosensors (such as Akt, PKC, Grb-2, GAP and Raf isoforms) that can monitor diverse signaling processes by their plasma membrane translocation or dissociation, and 3) an evanescent wave microscope setup to quantitatively monitor these plasma membrane translocation and dissociation events. In Phase I of the project, we will develop and test a 4 x 3 prototype cell array and, in Phase II, we will expand the cell array to 15 x 10. These two arrays will be used to simultaneously monitor different receptor or transformation induced signaling events in each of the 12, or 150, separate cell array segments on the same glass slide. While phase I includes a test of principle using existing biosensors and dominant negative and constitutively active interference proteins, Phase II will develop a library of such fluorescent translocation biosensors. Furthermore, we will develop two libraries of interference proteins by mutating a large number of serine/threonine kinases and small GTP-binding proteins into constitutively active and dominant negative constructs. As a test of the usefulness and the limitations of the ECAT method, we will determine the role of each member of these two interference libraries by testing them in the context of different receptor stimuli using the existing and newly developed fluorescent biosensors. Overall, this proposal will provide a new approach to signal transduction research and will provide a technological platform for the validation of cancer drug targets and the advancement of drug discovery.